Altered projection-specific synaptic remodeling and its modification by oxytocin in an idiopathic autism marmoset model

Alterations in the experience-dependent and autonomous elaboration of neural circuits are assumed to underlie autism spectrum disorder (ASD), though it is unclear what synaptic traits are responsible. Here, utilizing a valproic acid–induced ASD marmoset model, which shares common molecular features with idiopathic ASD, we investigate changes in the structural dynamics of tuft dendrites of upper-layer pyramidal neurons and adjacent axons in the dorsomedial prefrontal cortex through two-photon microscopy. In model marmosets, dendritic spine turnover is upregulated, and spines are generated in clusters and survived more often than in control marmosets. Presynaptic boutons in local axons, but not in commissural long-range axons, demonstrate hyperdynamic turnover in model marmosets, suggesting alterations in projection-specific plasticity. Intriguingly, nasal oxytocin administration attenuates clustered spine emergence in model marmosets. Enhanced clustered spine generation, possibly unique to certain presynaptic partners, may be associated with ASD and be a potential therapeutic target.

The authors report on the effects of valproic acid-treated marmosets in the dmPFC with a focus on dynamics of spine formation and removal, generation of clustered spines, and possible effects of nasal application of oxytocin on these parameters.I found the manuscript highly interesting and relevant for ASD research given the model system used, the techniques applied and the questions addressed.They report an increase in the synaptic dynamics of apical dendrites of L2/3 pyramidal neurons in the dmPFC, a brain area definitively associated with ASD.The turnover of dendritic spines was increased, in particular that of clustered spines.Overall this appears to be combined with an apparent increased survival of these spines which was dramatically higher compared to controls.This increase in spine clustering is reduced after nasal application of oxytocin.Technically the work is very challenging, and the longitudinal data of spine dynamics in the dmPFC using in vivo two-photon microscopy presented are of very high quality.The analysis of VPA-exposed adult marmosets is rather diverse from the typical study of ASD in the developing brain.Studies in the adult might have some principle differences and its use in ASD research is to me unclear but some investigations show indeed that there is plasticity in the ASD phenotype in adults.I am not sure of the relevance of a comparison of the spine generation rate and carry-over fraction of marmosets would have to be compared to a mouse model of ASD, here then MeCP2.Oxytocin receptors and circuits are prominently expressed in the VTA and have been shown in mouse ASD models to be important here in this region, while the authors look here in the dmPFC, and show also expression of these receptors.However, to me the effects here in the dmPFC of marmosets appears minute, and most parameters of spine dynamics appear not affected by oxytocin.It is also not clear whether these applications have any effect on ASD-relevant behaviour, so I am not convinced entirely whether these data should be part of the paper.The discussion is over-long and should be cut considerably.
Reviewer #2 (Remarks to the Author): By using in vivo time-lapse two-photon microscopy, Noguchi and colleagues examined structural spine dynamics of tuft dendrites of upper-layer pyramidal neurons and adjacent axons in the dorsomedial prefrontal cortex in adult marmosets.This manuscript reports that valproic acid (VPA)-induced adult ASD marmoset model exhibits upregulated dendritic spine turnover, increased new spine formation in clusters, enhanced new spine survivorship, and upregulated local axonal bouton dynamics.Using intranasal administration of oxytocin, the authors also demonstrated that oxytocin reduced the tendency of spines to cluster without affecting general turnover rate of spines in VPA model.The manuscript is well written, the experiments are well-designed, and obtained data are largely convincing.However, there are several important loose ends the authors should address and additional control experiments are needed.To strengthen their findings, the authors should address the concerns listed below: 1.The authors should show whether there is any difference in spine dynamics including spine formation, elimination, and new spine survivorship between 3-day (day 0 to day 3, or day 3 to day 6) vs 6-day (day 0 to day 6) observation experiments.It is not clear why the authors report 6-day data in 2. The authors mention that both male and female animals were used.Please clarify if there are any gender differences in the current results.Specifically, oxytocin and oxytocin receptor expressions are known to be sex-specific.
3. In Figure 2, the authors claim that spine clustering reached a plateau at a distance of 3 μm between spines, but it is not clear if this distance effect is abolished with clustering threshold > 3 μm.Please show spine clustering data with different thresholds such as 6 μm and 9 μm. 4. In Figure 2, please show the comparison of the % of clustered spine elimination (same as Fig. 2B but for elimination).Does the distance only affect clustered spine generation not elimination?Any crosstalk between formation and elimination was observed?Heterosynaptic mechanisms should be discussed (PMIDs: 37829671, 25558061).5.In Figure 3, please clarify the difference between carryover in panel C and spine stability of new spines in panel E. Are they conceptually the same observation?Please also show and compare the carryover and stability data from 3-day observation (short-term survivorship) vs 6-day observation (long-term survivorship).
6.Is axonal plasticity distance-dependent?In Figure 4, please show if presynaptic boutons also undergo dynamics in clusters similar to Figure 3. 7. What is the effect of nasal administration of oxytocin on spines in unexposed control marmosets?Can oxytocin alone induce spine dynamics, which might be abolished in VPA-exposed animals?Testing the physiological role of oxytocin in control marmosets is critical, as this is a big claim of the manuscript.In addition, there seems to be a trend towards a decrease in clustered spine generation and an increase in non-clustered spine generation.Are these experiments appropriately powered?What are the effects of oxytocin on survival fraction?Why are these clustered generation rates not being compared to UE and UE+OT?
Minor comments: 1. Please clarify their current VPA ASD marmoset model is induced by prenatal VPA exposure.This should be clearly shown in the experimental schema and timeline in Fig1A.In addition, the authors should discuss if their findings are specific to their method, in that only prenatal VPA, but not postnatal VPA exposure, leads to the observed spine and axon dynamics.
2. In figure 4, the authors show a change in the bouton density proportion between ipsi-and contralateral following VPA-induced ASD model.However, it is unclear from the text and graph, whether this is accompanied by a statistically significant increase of contralateral bouton density and a decrease in ipsilateral bouton density between groups.This may have interesting functional implications for microcircuit activity.
3. In figure 3, the authors provide evidence of an increased surviving fraction of newly generated spines.As a reader, one might expect that this increase in surviving fraction would result in an increase in spine density, yet this is not the case.This suggests the increase elimination compensates for the increase in surviving fraction.This is a very interesting finding; however, this raises a fundamental question on the biological impact of this discovery.What is the functional effect of this increase in turnover if it is not leading to an overall increase in spine density?Overall weaker (more new) synapses?Fewer/more functional synapses despite spine density?

Point-by-point responses to the reviewers:
We express our sincere gratitude to the reviewers for dedicating their valuable time to critically assess and comprehend the content of our paper.Their thoughtful review and understanding of our results report are greatly appreciated.

Reviewer #1 (Remarks to the Author):
The authors report on the effects of valproic acid-treated marmosets in the dmPFC with a focus on dynamics of spine formation and removal, generation of clustered spines, and possible effects of nasal application of oxytocin on these parameters.I found the manuscript highly interesting and relevant for ASD research given the model system used, the techniques applied and the questions addressed.
We thank Reviewer#1 for considering our results as both highly interesting and relevant for ASD research.
They report an increase in the synaptic dynamics of apical dendrites of L2/3 pyramidal neurons in the dmPFC, a brain area definitively associated with ASD.The turnover of dendritic spines was increased, in particular that of clustered spines.Overall this appears to be combined with an apparent increased survival of these spines which was dramatically higher compared to controls.This increase in spine clustering is reduced after nasal application of oxytocin.
Technically the work is very challenging, and the longitudinal data of spine dynamics in the dmPFC using in vivo two-photon microscopy presented are of very high quality.
The analysis of VPA-exposed adult marmosets is rather diverse from the typical study of ASD in the developing brain.Studies in the adult might have some principle differences and its use in ASD research is to me unclear but some investigations show indeed that there is plasticity in the ASD phenotype in adults.
1.The population of autistic adults is growing rapidly.They have multiple problems in their daily lives including anxiety, difficulties with verbal and nonverbal social communications, and sensory problems despite having undergone behavioral therapy and other interventions during their developmental years.
We believe it is desirable to have more treatment options for people after puberty in addition to those addressed in childhood.We add following sentences in the Introduction section: (L.76)A notable portion of individuals with ASD persists in experiencing a range of challenges such as anxiety or depression in their daily lives into adulthood.It is critical to understand ASD pathophysiology in adult ASD model animals and explore treatments.
I am not sure of the relevance of a comparison of the spine generation rate and carry-over fraction of marmosets would have to be compared to a mouse model of ASD, here then MeCP2.
2. We have considered the possibility that readers should want to know if there is a characteristic link between our marmoset model and an existing animal model (in this case, the mouse).However, the comparison with the specific mouse model may confuse the potential readers as Reviewer #1 suggested.Therefore, we would like to remove previous Figures S2B and S2C  Oxytocin receptors and circuits are prominently expressed in the VTA and have been shown in mouse ASD models to be important here in this region, while the authors look here in the dmPFC, and show also expression of these receptors.However, to me the effects here in the dmPFC of marmosets appears minute, and most parameters of spine dynamics appear not affected by oxytocin.It is also not clear whether these applications have any effect on ASD-relevant behaviour, so I am not convinced entirely whether these data should be part of the paper.
The discussion is over-long and should be cut considerably.
3. As noted by Reviewer #1, it is known that oxytocin receptors are strongly expressed including VTA, basal ganglia, nucleus accumbens, and hypothalamus, while their expression in the cortex is relatively low.Therefore, it is possible that indirect effects from those nuclei may be exerted on the cortex.On the other hand, oxytocin receptors in the prefrontal cortex are detectable and may play a role (Fig. S6).
As shown in the new Fig. 5, oxytocin also altered the bias of clustered dendritic spine generation in UE marmosets.(but in the opposite direction from VPA-exposed animals).This finding underscores the conclusion that oxytocin administration induces changes in synaptic properties within the prefrontal cortex.
In response to the suggestion of Reviewer #1, we have revised the Discussion section for conciseness.Specifically, the following statements were deleted (displayed by the line number of the previous manuscript): (L. 328) Clustered emergent spines that receive structurally or functionally similar inputs are thought to enable nonlinear dendritic computation and may facilitate the ability to acquire memory and behavioral skills through learning and experience 11, 12, 14-17, 67 .
Alternatively, clustered generated spines may also help stabilize formed circuits and accelerate learning due to their redundant involvement in the circuit 68 .In fact, (L.347) Compared to VPA-exposed marmosets, the degree of clustered emergence in MeCP2 duplication mice appears to be substantially lower.The reasons for this discrepancy are currently unclear.However, it may be due to differences in spine density, plasticity molecules, or subcellular organelles between species, cortical areas, syndromic and idiopathic ASD models, and deep and upper-layer pyramidal neurons.In any case, the (L.406) Suppression of excessive microglial activation with minocycline or other matrix metalloproteinase-9 inhibitors has been reported to suppress excessive spine turnover in fragile X model mice 8 .Our transcriptomic analysis also revealed significant downregulation of myelin-related genes, such as PLP1, which suppresses heightened cortical plasticity (see Supplementary Data 1).On the other hand, it has been shown that the stress hormone corticosterone increases spine turnover in mice 81 .

Reviewer #2 (Remarks to the Author):
By using in vivo time-lapse two-photon microscopy, Noguchi and colleagues examined structural spine dynamics of tuft dendrites of upper-layer pyramidal neurons and adjacent axons in the dorsomedial prefrontal cortex in adult marmosets.This manuscript reports that valproic acid (VPA)induced adult ASD marmoset model exhibits upregulated dendritic spine turnover, increased new spine formation in clusters, enhanced new spine survivorship, and upregulated local axonal bouton dynamics.Using intranasal administration of oxytocin, the authors also demonstrated that oxytocin reduced the tendency of spines to cluster without affecting general turnover rate of spines in VPA model.The manuscript is well written, the experiments are well-designed, and obtained data are largely convincing.However, there are several important loose ends the authors should address and additional control experiments are needed.To strengthen their findings, the authors should address the concerns listed below: We thank Reviewer #2 for dedicating time to thoroughly review our manuscript.We are deeply honored to have received not only a comprehensive understanding of the manuscript but also a positive evaluation of its experimental design and the results derived from it.

Major comments:
1.The authors should show whether there is any difference in spine dynamics including spine formation, elimination, and new spine survivorship between 3-day (day 0 to day 3, or day 3 to day 6) vs 6-day (day 0 to day 6) observation experiments.It is not clear why the authors report 6-day data in 1.For the previous Figures 1F and 1G, a 6-day observation period was initially employed, while a 3day period was utilized for the remaining figures.Nevertheless, recognizing the potential confounding effects of mixed observation periods on figure interpretation, we harmonized the observation period to 3 days.Consequently, our analysis yielded essentially identical results to those obtained during the 6-day period (Revised Figs.1F and 1G).
2. The authors mention that both male and female animals were used.Please clarify if there are any gender differences in the current results.Specifically, oxytocin and oxytocin receptor expressions are known to be sex-specific.
2. In reference to Figure 5, we analyzed whether there were sex differences in dendritic spine generation or elimination following oxytocin administration to VPA-exposed animals (Supplementary Figures S9-S12).The scatterplot data for each dendrite shown separately for UE and VPA-exposed males and females.As a result, we could not detect statistically significant sex differences.
We modified the manuscript as follows: (L. 303) It is known that there is sexual dimorphism in oxytocin receptor expression that well investigated in rodents.On the other hand, it has been reported that an absence of sex differences in OT-immunoreactive neurons in marmoset brain regions such as paraventricularis and supraopticus of the hypothalamus as well as in the bed nucleus of the stria terminalis and the medial amygdala.We therefore examined the effect of sexual differences on the effects of oxytocin by displaying scatter plots of the generation or elimination rate of spines before and after oxytocin administration (Supplementary Figure S9-S12).The scatterplot data for each dendrite shown separately for UE and VPA-exposed males and females.We could not detect statistically significant sex differences in VPA-exposed marmosets with respect to total, clustered, and non-clustered spine generation/elimination, potentially due to the considerable variation in values.Similarly, in the UE marmosets, although we detected statistically significant sex differences in several items (total and clustered spine generation at D6-9, and total spine elimination at D0-3), we could not find any systematically comprehensible sex differences across the parameters under investigation.Furthermore, in the Discussion Limitations section, we stated the following: (L.455) We could not conclude a systematic sex difference in the present data (depicted in Figs.

S11-S14 for oxytocin treatment).
3. In Figure 2, the authors claim that spine clustering reached a plateau at a distance of 3 µm between spines, but it is not clear if this distance effect is abolished with clustering threshold > 3 µm.Please show spine clustering data with different thresholds such as 6 µm and 9 µm.
3. We revised Supplementary Fig. S2 and main text as follows: (L. 164) We explored the impact of adjusting cluster thresholds to 3, 6, and 9 microns on the outcomes of spine generation and elimination clustering (Figure S2C-P).The clustering probability of the generated spines actually observed in VPA-exposed animals corresponded to a probability of P = 0.00042 for the distribution of clustering probabilities predicted by the simulation at the 3 µm threshold (Figure S2D).This P-value is much smaller than the probabilities at the 6 µm (P = 0.015) or 9 µm (P = 0.25) thresholds (Figures S2F and S2H).To investigate this further, we plotted the actual clustering probabilities against the simulated clustering probability, and again found that the difference between chance-level clustering and observed clustering was greatest at the 3 µm threshold that provides the Youden's index in the chart (Fig. S2O) 39 .Since the specific clustering bias disappears at the 9 µm threshold, indicating a physiological significance of the interaction within this distance.This analysis elucidates that the threshold set at 3 µm offers a good cut-off value for assessing spine clustered generation.In contrast, the clustering probability of the generated spine pairs was remained below the 95th percentile in the UE group (Figure 2E). 4. In Figure 2, please show the comparison of the % of clustered spine elimination (same as Fig. 2B but for elimination).Does the distance only affect clustered spine generation not elimination?Any crosstalk between formation and elimination was observed?Heterosynaptic mechanisms should be discussed (PMIDs: 37829671, 25558061).4. We conducted an analysis of clustered spine elimination and prepared a revised Fig. S3 that incorporates the previous Fig.2G and H.Although there is a disparity in the effect of distance on clustered spine elimination between UE and VPA-exposed animals (reviced Fig. S3C), this effect was considerably less pronounced than spine generation (Fig. 2C): (L. 177) On the other hand, the specific clustering of eliminated spines was not as prominent as generated spines either in UE and VPA-exposed animals (Figures S2I-N, S2P, and S3).
Although there is a disparity in the effect of distance on clustered spine elimination between UE and VPA-exposed animals (Figure S3C), this effect was considerably less pronounced than spine generation (Figure S2B).The slight increase in clustered elimination observed in VPAexposed marmosets could stem from heightened nonspecific clustering associated with the larger number of eliminated spines.Simulation analysis revealed that the clustering bias of spine elimination in VPA-exposed animals is not significant (Figures S2P, S3D, and S3E).
We modified the main text about the crosstalk between spine formation and elimination as follows: (L. 186) Furthermore, an analysis of the crosstalk between spine generation and elimination was conducted, as depicted in Figure S4.Cumulative frequency distributions were generated for the distances between newly generated spines and their nearest eliminating spines.
Consequently, no statistically significant difference was observed between the UE and VPAexposed marmosets in this regard (Figure S4).
Regarding the heterosynaptic mechanism identified in the present experimental setup, we have incorporated the following sentences into the Discussion section: (L.376) There are several studies that have shown that synaptic plasticity diffuses from stimulated spines into neighboring spines and interacts there 5,34,39,[63][64][65][66] .The relationship between heterosynaptic plasticity and the subsequent new generation or elimination of surrounding spines is a subject for future work. 5.In Figure 3, please clarify the difference between carryover in panel C and spine stability of new spines in panel E. Are they conceptually the same observation?Please also show and compare the carryover and stability data from 3-day observation (short-term survivorship) vs 6-day observation (long-term survivorship).
5. We revised Figure 3 and main text to clarify the difference between carryover and spine stability (spine "stability" will be referred to as "survival rate" hereafter) of new spines: (L. 200) The carryover spines were defined as the newly generated spines that survived to the last session.In other words, carryover spines are spines whose presence was denied by the first observation and confirmed by the second and third observations (Figures 3A and 3B, GS).As discussed below, the survival rate of carryover spines is not different between UE and VPAexposed animals.However, since the fraction of newly generated spines are two times higher in VPA-exposed animals (Figure 1D), the fraction of carryover spines was two times higher in VPA-exposed marmosets than in UE marmosets (Figure 3C).(L.214) By calculating the carryover fraction, we can understand how newly generated spines contribute to the overall synaptic population.Next, by calculating the spine survival rate, we can focus on the persistence of each newly generated and pre-existing spine, offering a more targeted measure of synaptic resilience.Spine survival rate was defined as the percentage of spines present at the second observation that were still present at the third observation (Figure 3B)."Carryover spines" are spines whose presence was denied by the first two-photon microscopy observation and confirmed by the second and third observations.Therefore, we cannot show longterm (6-day) survivorship of the carryover spines.6.Is axonal plasticity distance-dependent?In Figure 4, please show if presynaptic boutons also undergo dynamics in clusters similar to Figure 3. 6. Due to the considerably lower density of boutons compared to dendritic spines in the sample utilized for this investigation, we observed a relatively limited occurrence of simultaneous bouton generation or elimination within the measured axons.Consequently, it was not possible to create a cumulative distribution with a high degree of confidence (N = 6, 6, 5, and 10 axons featuring simultaneously generated bouton pairs, and N = 5, 1, 4, and 5 axons featuring simultaneously eliminated bouton pairs, out of a total of 28, 21, 22, and 25 UE-ipsi, UE-contra, VPA-ipsi, and VPA-contra axons, respectively).Consequently, we refrain from incorporating this data into the main text.We added the following to the Results section: (L.307) Due to the lack of enough coincident events, we were unable to analyze the interactions between newly generated or eliminated axonal boutons.
7. What is the effect of nasal administration of oxytocin on spines in unexposed control marmosets?Can oxytocin alone induce spine dynamics, which might be abolished in VPA-exposed animals?
Testing the physiological role of oxytocin in control marmosets is critical, as this is a big claim of the manuscript.In addition, there seems to be a trend towards a decrease in clustered spine generation and an increase in non-clustered spine generation.Are these experiments appropriately powered?What are the effects of oxytocin on survival fraction?Why are these clustered generation rates not being compared to UE and UE+OT? 7. We express our gratitude for the opportunity to investigate the effects of oxytocin administration on the clustered generation or elimination of dendritic spines in UE marmosets.As demonstrated in the revised Fig. 5, our analyses indicate that oxytocin administration induces alterations in clustered spine generation even in UE marmosets.Notably, oxytocin tended to promote clustered spine generation, contrary to the case in VPA-exposed animals.The underlying mechanism remains elusive and warrants further investigation, suggesting that synaptic function is quite different in UE and VPAexposed animals.
Regarding the statistical power to detect clustered spine generation or elimination, it is pertinent to note the substantial variability present within each dendrite, encompassing instances where no clusters are observed.Consequently, we were unable to discern statistically significant changes in the average occurrence of clustered spine generation (Fig. 5C) in either UE or VPA-exposed animals.To address this limitation, we employed simulation analysis utilizing a model that factors in dendrite length and the number of spines, offering enhanced statistical power (refer to Figure 5E-K and Figure S7D-J).In relation to the above, we modified the Results section (L.290~302) accordingly.

Minor comments:
1. Please clarify their current VPA ASD marmoset model is induced by prenatal VPA exposure.This should be clearly shown in the experimental schema and timeline in Fig1A.In addition, the authors should discuss if their findings are specific to their method, in that only prenatal VPA, but not postnatal VPA exposure, leads to the observed spine and axon dynamics.A change was made to the color and shape of the symbols to ensure that the figures are clearly identifiable when printed in black and white, as well as being understandable by users with color blindness.
To number the supplementary figures in the order of appearance in the main text, Supplementary Figures 8, 9, 10, 11.and 12 are renumbered as Supplementary Fig. 12, 8, 9, 10, and 11, respectively.Source data for Supplementary Figure 6 are provided.
Fig 1 and 3-day results in other figures.Can authors conclude the same findings in Fig1 by using 3-day data only and/or in Fig 3-5 by 6-day data only?
and the following sentences in the main text: (L.199 of previous manuscript version) Lastly, we compared the clustering of VPA-exposed marmosets with those of a mouse model.…Thus, the rate of spine clustering in the marmoset model differed markedly in comparison to the MeCP2 duplication mouse model.
strong clustering tendency in the dmPFC of VPA-exposed marmosets underscores the importance of clustering in the pathogenesis of idiopathic ASD and warrants further research of clustered spine emergence in both physiological and pathophysiological conditions.(L.375) It will be interesting to analyze whether mismatches in plasticity control involving local and long-range connections facilitate autonomous local circuit remodeling independent of the highly contextualized information computed in the brain-wide global network.(L.389) Oxytocin receptors in marmosets are notably expressed in the basal ganglia of the nucleus basalis of Meynert (NBM), a prominent cholinergic region within the brain.The NBM serves as a significant source of cholinergic innervation to various brain regions, including the neocortex, and plays a pivotal role in regulating selective attention and motivational processes 54 .It is plausible that the alteration of spine clustering induced by oxytocin may involve the modulation of cholinergic inputs.Oxytocin's inhibitory effect on spine clustering could restrain excessively prolonged circuit stability and mitigate behavioral perseverance, as reported in human ASD 78 .

Fig 1 and 3 -
Fig 1 and 3-day results in other figures.Can authors conclude the same findings in Fig1 by using 3day data only and/or in Fig 3-5 by 6-day data only?

Fig. 1A :
Fig. 1A: VPA exposure was added to the experimental time course.

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Fig. 1C, D: Individual data points were added to the bar charts.

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Fig. 1F, G: Observation period was changed to 3-day.

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Fig. 3C-G: Graph titles were added.Graph y-axis labels were changed.

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Fig.3E, F: Individual data points were added to the bar charts.

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Fig. 4D, F, G, H: Individual data points were added to the bar charts.

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Fig. 5B-D: Statistical analysis method was changed to 2-Way ANOVA.

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Fig. 5B-D: Individual data points were added to the bar charts.

Figures and Supplementary figures
Figures and Supplementary figures: Figures and Supplementary figures:

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Figure numbering labels have been modified from upper case letters to lower case letters.(Figures and main text) )